Agarose Gel Protocol

Assembling Electrophoresis Cell

  • Gather all parts. These should be located on the shelf above the pipette rack, but can often be found in/near the sinks or in the 4oC : Base, Gel-Casting Gates (two), Gel Tray,Comb(s),Safety Lid with Electrical Cables
  • Place the base on an open part of the counter
  • If base is not level, adjust Leveling Feet accordingly
  • Insert the Gel-Casting Gates into their corresponding grooves
  • Place the Gel Tray between the Gel-Casting Gates
  • NOTE: there is a nice diagram in the BioRad Life Science Research Products catalog on page 255

Preparing the Gel

  • Combine either 40 mL buffer with 400 mg agarose in an Erlenmeyer flask for a small gel or at least 100 mL buffer with 1.0 g agarose, to create a 1% solution
  • Use 1x TAE as your buffer if your samples are DNA extracts, and 1x TBE if your samples are PCR amplified DNA
  • Plug up flask with a paper towel
  • Microwave flask for roughly one to two minutes for a small gel and two to three minutes for a large gel to dissolve agarose, stopping microwave and swirling flask every time it shows signs of boiling
  • Once completely dissolved, cool solution under running water until the flask can be held without burning skin
  • Double check that Gel-Casting Gates are securely in place and pour agarose solution onto Gel Tray
  • For small gels, pour all 40 mL; for large gels, fill between two and three ticks (these are on the sides of the base)
  • Rinse out Erlenmeyer flask, fill with water, and set aside
  • Pop any bubbles in gel with a syringe needle
  • Insert appropriate Comb(s) into the appropriate Comb Slots
  • Allow gel to solidify
  • Thoroughly clean out Erlenmeyer flask
  • Gel can now be saved for later use. Place in Ziplock bag and store at 4oC

Loading Samples

  • Remove Comb(s) and Gel-Casting Gates
  • Wipe gel off Comb(s) and Gel-Casting Gates with paper towel to remove any agarose residue
  • Fill base with same buffer as above to cover gel
  • On a strip of Parafilm, measure out 5 µL sample and 2 µL Nucleic Acid Sample Loading Buffer, 5x
  • Mix sample and loading buffer by slowly sucking in and out of pipettor tip
  • Load samples into wells
  • Never use outside lanes
  • Dip pipette tip into buffer on side with red Bananna Plug/Electrode Cassette before loading well to remove any excess dye
  • Carefully lower tip half-way into well, being careful not to touch gel
  • Load well slowly to ensuring none of the sample escapes into the buffer
  • Raise pipette tip out of buffer before releasing thumb (very important)
  • Load 5 µL Precision Molecular Mass Standard into one well and 1 µL into another

Running Gel

  • Place Safety Lid on Base, matching up Electrical Cables to Bananna Plugs/Electrode Cassettes according to color
  • Plug Electrical Cables into power source (PowerPack 300 or EC105)
  • Turn on power source, set voltage to 110V for small gels and 120V for large gels, and press the “running man” button to begin (verify that the gel is running by looking for a stream of bubbles originating at the black Bananna Plug/Electrode Cassette)
  • Run small gels for approximately 20 minutes and large ones until the dye is 75% across the gel
  • Turn off and unplug power source, and disconnect Electrical Cables
  • Remove Safety Lid and Comb(s)
  • Remove Gel Tray with gel on it (gel can either be imaged or stored in a Ziplock bag at 4oC)
  • Pour buffer out of Base into a labeled container and store at 4oC for later use
  • Clean out Base, Comb(s), and Gel Tray

Imaging Gel and Quantifying Bands

  • Follow standard procedure